AEEC INFECTIONS Informe resumido

In vivo and In vitro stability of the LEE: the pathogenicity island of AEEC strain

The objective of this WP was to quantify and analyse in vivo and in vitro, the stability of the LEE in AEEC strains of serotype O103:H2. The specific aim is to introduce a tetAR(B) cassette marker in the LEE and take the advantage of a simple one-step selection for Tc-sensitive revertants. We further characterized the stability of the LEE in a representative collection of AEEC with LEE inserted in different tRNA.

Major finding during the project:In contrast to other E. coli pathogenicity islands, the LEE was supposed to be stable. But preliminary observations obtained in our laboratory suggested that the LEE was very likely unstable when AEEC strains of serotype O103:H2 were used to infect orally rabbits. Therefore, our objective was to confirm this result and to quantify in vivo and in vitro the stability of the LEE in two AEEC strains of serotype O103:H2, the rabbit EPEC strain E22 and the human EHEC strain PMK5. These strains produce b1 and e intimin subtypes respectively.

A major limitation to study the stability of the LEE of AEEC strains was the difficulty to detect deletions of the PAI. Therefore, we have developed a strategy based on the island probing, a method used to identify the Shigella flexneri she PAI. We have been able to isolate several Tc-sensitive derivatives of E22 in which the LEE was excised. In contrast, Tc-sensitive revertant clones of PMK5 were never obtained. In EPEC strain, the frequency of excision of the LEE was about 1.10-6 (corresponding to the number of Tc-sensitive clones relative to the total population plated onto LB medium containing kanamycin). We found no the effects of culture conditions (temperature, osmolarity, etc.) on excision of the LEE in vitro.

We have then orally infected 10 rabbits with E22 tir::tetAR, lacZ::km. The inoculated strain was recovered from the rabbits by plating dilutions of the faeces onto MacConkey medium with Kanamycin. Instability of the LEE was estimated by plating the Km+ Lac - derivatives onto fusaric acid and kanamycin medium in order to select Tc-sensitive revertants. The method is based on the finding that Tc-resistant bacteria are hypersensitive to lipophilic chelating agents such as fusaric acid. Using this method, we have been able to confirm that the LEE was also unstable. The frequency of the appearance of Tc-sensitive revertants was estimated to less than 10-6. Of note we have been able to isolate these revertants in only two rabbits out of ten.